NRF2 Screening Assays and Related Methods and Compositions

Abstract

Provided are certain methods of screening, identifying, and evaluating neuroprotective compounds useful for treatment of neurological diseases, such as, e.g., multiple sclerosis (MS). The compounds described upregulate the cellular cytoprotective pathway regulated by Nrf2. Also provided are certain methods of utilizing such compounds in therapy for neurological disease, particularly, for slowing or reducing demyelination, axonal loss, or neuronal and oligodendrocyte death.

1 . A method of evaluating neuroprotective properties of at least one test compound, the method comprising:
 a) contacting a cell with the at least one test compound,   b) determining whether the Nrf2 pathway is upregulated in the cell,   c) determining whether the at least one test compound slows or prevents at least one of demyelination, axonal loss, and neuronal death, and   d) selecting the test compound as a candidate for treating neurodegeneration in a neurological disease if 1) the Nrf2 pathway is upregulated and, optionally,   e) further determining whether at least one of demyelination, axonal loss, and neuronal death are prevented or slowed by the compound.   
     
     
         2 . The method of  claim 1 , wherein the neurological disease is a demyelinating disease. 
     
     
         3 . The method of  claim 2 , wherein the demyelinating disease is multiple sclerosis. 
     
     
         4 . The method of  claim 1 , wherein the cell in step a) is contacted with a plurality of test compounds. 
     
     
         5 . The method of  claim 1 , wherein the Nrf2 pathway upregulation is determined by the levels of expression or activity of NQO1. 
     
     
         6 . The method of  claim 1 , wherein the Nrf2 pathway is upregulated by at least 30% as indicated by one or more of the following parameters:
 i) expression levels of at least one of endogenously produced and exogenously introduced Nrf2;   ii) at least one of subcellular localization and nuclear translocation of Nrf2;   iii) the expression level of at least one gene under control of Nrf2;   iv) the level of Nrf2 binding to the Nrf2-binding DNA element ARE;   v) the stability of Nrf2/Keap1 complexes; and   vi) modification levels of at least one protein selected from Keap1 and other Nrf2/Keap1-associated proteins.   
     
     
         7 . The method of  claim 6 , wherein the at least one gene under control of Nrf2 is an Nrf2-regulated reporter gene in an artificial reporter construct. 
     
     
         8 . The method of  claim 1 , further comprising comparing the level of Nrf2 pathway upregulation by the at least one test compound with the level of Nrf2 pathway upregulation by at least one comparator compound. 
     
     
         9 . The method of  claim 8 , wherein the at least one comparator compound is selected from dimethyl fumarate and monomethyl fumarate. 
     
     
         10 . The method of  claim 1 , wherein the at least one test compound has the structure of Formula I: 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein: 
         X is O; S; C(R)(C 1-12 )alkyl; or C(R)(C 2-12 )alkenyl, wherein R is H, (C 1-12 )alkyl or (C 2-12 )alkenyl; 
         R 1 , R 2 , R 3  and R 4  are independently selected from H; OH; O − ; CO 2 H; CO 2   − ; SH; S − ; SO 2 H; SO 2   − ; (C 1-24 )alkyl; (C1-24)alkenyl; (C 6-50 )aryl; CO 2 (C 1-24 )alkyl; SO 2 (C 1-24 )alkyl; CO 2 (C 1-24 )alkenyl; SO 2 (C 1-24 )alkenyl; CO 2 Y, wherein Y is psoralen-9-yl, retinyl, alpha-tocopherol, calciferyl, corticosteroid-21-yl, or monosaccarid-ω-yl; (C 1-24 )alkoxy; (C 1-24 )alkenyloxy; (C 6-50 )aryloxy; (C 1-24 )alkylthio; (C 1-24 )alkenylthio; (C 6-50 )arylthio; amino; amido; arylalkyl; cyano; nitro; sulfonyl; sulfoxide; sulfonamide; formyl; keto; and D and L natural or unnatural amino acids; wherein any two of X, R 1 , R 2 , R 3 , and R 4  are optionally joined to form a cyclic moiety; and 
         wherein the alkyl, alkoxy, alkenyl, alkenyloxy, aryl and aryloxy is optionally substituted with at least one group chosen from halogen, OH, (C 1-4 )alkoxy, nitro and cyano. 
       
     
     
         11 . The method of  claim 1 , wherein the at least one test compound is selected from fumaric acid, its salts, and fumaric acid derivatives. 
     
     
         12 . The method of  claim 1 , wherein the at least one test compound is chosen from FTY 720, ABT-874, GSK683699, NeuroVax, MLN 1202, interferon γ, Tysabri™, Rituxan, TV 5010, NBI-788, MBP8298, Cladribine, Teriflunomide, Temsirolimus, and Laquinimod. 
     
     
         13 . A method of treating a mammal having a neurological disease, comprising:
 a) selecting a test compound according to the method of  claim 1 , and   b) administering the selected test compound to a mammal in need thereof, thereby treating neurodegeneration in the mammal.   
     
     
         14 . A method of treating a mammal having a neurological disease by combination therapy, the method comprising:
 a) administering to the mammal a therapeutically effective amount of at least one first compound that upregulates the Nrf2 pathway, and   b) administering a therapeutically effective amount of at least one second compound that does not upregulate the Nrf2 pathway.   
     
     
         15 . The method of  claim 14 , wherein the at least one first compound is selected from fumaric acid, its salts, and fumaric acid derivatives. 
     
     
         16 . The method of  claim 14 , wherein the neurological disease is a demyelinating disease. 
     
     
         17 . The method of  claim 16 , wherein the demyelinating disease is multiple sclerosis. 
     
     
         18 . A method of treating amyotrophic lateral sclerosis (ALS), comprising treating a subject in need of such treatment with a therapeutically effective amount of dimethyl fumarate (DMF), monomethyl fumarate (MMF), or a combination thereof. 
     
     
         19 . The method of  claim 18 , wherein the therapeutically effective amount of DMF, MMF, or a combination thereof is from about 200 mg to about 800 mg per day. 
     
     
         20 - 24 . (canceled) 
     
     
         25 . A method of treating amyotrophic lateral sclerosis (ALS), comprising orally administering to a subject in need of such treatment a therapeutically effective amount of DMF. 
     
     
         26 - 30 . (canceled)